Phosphorylation-dependent Changes in the R-region Interactions Contribute to Regulation of the CFTR Chloride Channel
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Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the defective protein in cystic fibrosis, is an ion channel regulated by PKA and PKC phosphorylation of its regulatory region (R-region) through a mechanism still not completely understood, preventing a full comprehension of the process involved in CFTR activation. To provide novel insights into the CFTR activation mechanism, I analyzed in situ and in vitro interactions of the R-region with other parts of CFTR at different phosphorylation conditions (PKA, PKC, PKA+PKC). For in situ analysis, I used a split-CFTR construct expressing the front half (FH;N-tail/TMD1/NBD1), back half (BH;TMD2/NBD2/C-tail) and the R-region as three separate polypeptides expressed in BHK cells. I found that PKA stimulation increased both FH-R and BH-R interactions and PKC+PKA further increased only FH-R interactions whereas PKC stimulation alone had no effect. Inactivation of PKC consensus site S686 (S686A) significantly reduced basal BH-R interaction and prevented the further enhancing of FH-R interactions by PKC+PKA phosphorylation that was found with the wild-type R-region. The opposite phosphomimetic mutation S686D restored basal BH-R and rescued 80% of FH-R interactions after PKC+PKA stimulation compared to wild-type levels. As the channel function is mainly stimulated by PKA phosphorylation of the R region, and this response is doubled by PKC+PKA phosphorylation, results from this thesis suggest that PKC enhances PKA effect by increasing R-region interactions with the FH. Also, PKC site S686 was found to be crucial for the PKC enhancing effect which appeared to be mediated by a permissive interaction of the R-region with the BH, allowing FH-R interactions to be enhanced by PKC. Analysis of in vitro interactions of the R-region with six polypeptides corresponding to the N- and C-terminal tails and the four cytoplasmic loops (CLs) of CFTR showed that phosphorylation strongly increased R-region interactions with both N- and C-tails of CFTR. Moreover, PKA and PKC+PKA phosphorylation reduced R-region interactions with CLs 4 and 3, respectively, a mechanism that permits the channel activation by PKA and PKC enhancing effect. All together, these results demonstrate that phosphorylation induces an R-region shift from the cytoplasmic loops to both amino and carboxyl ends of the protein, to activate the channel.